KR20110033943A - Selection and use of lactic acid bacteria for reducing inflammmation caused by helicobacter - Google Patents
Selection and use of lactic acid bacteria for reducing inflammmation caused by helicobacter Download PDFInfo
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- KR20110033943A KR20110033943A KR1020117004118A KR20117004118A KR20110033943A KR 20110033943 A KR20110033943 A KR 20110033943A KR 1020117004118 A KR1020117004118 A KR 1020117004118A KR 20117004118 A KR20117004118 A KR 20117004118A KR 20110033943 A KR20110033943 A KR 20110033943A
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Abstract
본 발명은 위장 염증, 예를 들면, 헬리코박터 파일로리(Helicobacteri pylori)에 기인한 위장 염증을 감소시키는 능력에 기초하여 선별된 특정 락토바실러스(Lactobacillus) 균주 및 이런 균주를 함유하는 산물에 관하고, 또한 사람 투여용으로 헬리코박터 파일로리(Helicobacteri pylori)와 연관된 염증의 치료 또는 예방을 위한 약품, 상기 선별된 균주가 성장하는 조정 배지, 이런 조정 배지의 단백질-함유 추출물에 관한다.The present invention relates to certain Lactobacillus strains and products containing these strains, selected on the basis of their ability to reduce gastrointestinal inflammation, for example, gastrointestinal inflammation due to Helicobacteri pylori , and also humans. relates to extracts containing-conditioned medium, the conditioned media of this protein to the drug, the selected strains for the treatment or prevention of inflammation associated with pylori (Helicobacteri pylori) for administration Helicobacter growth.
Description
본 발명은 비-병원성 소염 박테리아 균주를 선별하는 방법 및 위장 박테리아, 예를 들면, 헬리코박터 파일로리(Helicobacteri pylori), 헬리코박터(Helicobacter)의 다른 종 및 다른 염증-유발 위장 병원균에 의해 유발되는 염증의 치료와 예방을 위하여 이런 균주를 이용하는 산물과 방법에 관한다.The present invention relates to methods for screening non-pathogenic anti-inflammatory bacterial strains and to treatment of inflammation caused by gastrointestinal bacteria such as Helicobacteri pylori , other species of Helicobacter and other inflammation-induced gastrointestinal pathogens. It relates to products and methods of using these strains for prevention.
헬리코박터 파일로리(Helicobacteri pylori)는 특히, 위에서 산을 중화시키는 우레아제(urease)를 생산하는 능력으로 인하여 위에 군총을 형성하는 나선형 박테리아이다. 우레아제는 위에 풍부하게 존재하는 요소를 강염기인 중탄산염과 암모니아로 변환시킨다. 이는 헬리코박터 파일로리(H. pylori) 세포 주변에 대량의 산-중화 염기를 발생시켜 위산으로부터 이들을 보호한다. 헬리코박터 파일로리(H. pylori) 세포는 위 점액층에 침투하고 횡단하며 위의 내막에서 상피 세포에 부착한다. 헬리코박터 파일로리(H. pylori)의 적어도 일부 균주는 독소를 생산하는 능력을 보유한다. 헬리코박터 파일로리(H. pylori)에 감염되면 면역계가 활성화되어 백혈구 세포, 킬러 T 세포 및 다른 감염-저해제가 감염 부위에 동원되지만, 신체의 면역계는 위의 점액 내막에서 헬리코박터 파일로리(H. pylori)의 효과를 반전시키지 못한다. 헬리코박터 파일로리(H. pylori) 세포는 내막에 존속하고, 면역계는 이들 세포에 대한 반응을 고조시켜 충분한 소염 기전이 작동되지 않으면 염증이 발생하게 된다. 헬리코박터 파일로리(H. pylori) 감염동안, 숙주 상피 수상돌기 세포, 자연 킬러 세포, T-세포 및 다른 면역 방어 세포에 의해 생성된 사이토킨 세포내 신호 단백질은 침입 병원균에 대한 면역 반응을 전파한다. 결과적으로, 숙주 호중구가 위 상피에 유인되어 침투하고 전체 감염 기간 동안 이곳에 존속한다. 이들 세포는 특히, 반응성 산소 산물, 예를 들면, 슈퍼옥사이드 라디칼(superoxide radical)을 생성하는데, 이들은 상피 세포에서 산화를 유발하고, 결과적으로 상피 세포 사멸, 궤양 형성, 암발생을 초래한다. 또한, 헬리코박터 파일로리(H. pylori)는 영양 공급원을 제공하는 위 상피에서 숙주로부터 영양분을 누출시켜 헬리코박터 파일로리(H. pylori) 세포를 유지하고 감염 및 이의 결과를 악화시킨다. 헬리코박터 파일로리(H. pylori)는 숙주의 면역 반응에도 불구하고 사람 면역계를 회피하고 위에서 생존할 수 있는데, 이런 회피 기전은 현재 연구 과제중의 하나이다.Helicobacter pylori (Helicobacteri pylori) is a spiral bacteria that forms a gunchong over due to the ability to produce urease (urease) to neutralize the acid from above, in particular. Urease converts urea, which is abundant in the stomach, into strong bases bicarbonate and ammonia. This generates large amounts of acid-neutralizing bases around H. pylori cells and protects them from gastric acid. Helicobacter pylori (H. pylori) cells infiltrate the above jeomaekcheung crossing, and attached to the epithelial cells from the lining of the stomach. At least some strains of H. pylori retain the ability to produce toxins. Infection with H. pylori activates the immune system so that leukocytes, killer T cells, and other infection-inhibiting agents are recruited to the site of infection, but the body's immune system is responsible for the effects of H. pylori in the mucous membrane of the stomach. Cannot reverse. Helicobacter pylori (H. pylori) cells are retained in the inner film, and if the immune system is to heighten the response in the cells sufficient anti-inflammatory mechanisms are not operating is generated inflammation. During H. pylori infection, cytokine intracellular signal proteins produced by host epithelial dendritic cells, natural killer cells, T-cells and other immune defense cells propagate the immune response against invading pathogens. As a result, host neutrophils are attracted to and infiltrate the gastric epithelium and remain there for the entire infection period. These cells produce, in particular, reactive oxygen products, such as superoxide radicals, which cause oxidation in epithelial cells, resulting in epithelial cell death, ulcer formation, and cancer development. Further, the Helicobacter pylori (H. pylori) is maintained by the leakage of nutrients from the host in the epithelium above to provide a nutritional source of Helicobacter pylori (H. pylori) cells and exacerbate the infection and its results. Helicobacter pylori ( H. pylori ) can evade the human immune system and survive in the stomach despite the host's immune response. This avoidance mechanism is one of the current research challenges.
현재의 요법은 본 발명의 목적인 충분한 소염 기전을 가능하게 하는 방법과 같은 헬리코박터 파일로리(H. pylori) 감염에 대한 숙주의 과도한 면역 반응 효과를 제거하려는 시도보다는 항생제와 양성자 펌프 저해물질을 통하여 헬리코박터 파일로리(H. pylori)를 박멸하는데 기초한다.Current therapies are directed to Helicobacter pylori through antibiotics and proton pump inhibitors, rather than attempting to eliminate the effects of the host's excessive immune response to H. pylori infection, such as methods that enable sufficient anti-inflammatory mechanisms for the purposes of the present invention. H. pylori ).
따라서, 헬리코박터 파일로리(H. pylori) 감염은 소화성 궤양(peptic ulcer), 위암, 위 점막-결합된 림프 조직 림프종을 비롯하여 위염, 위궤양, 십이지장궤양의 발병 위험을 증가시킨다. 이들 문제는 헬리코박터 파일로리(H. pylori) 세포에 의해 직접 유도되지는 않지만, 헬리코박터 파일로리(H. pylori)에 대한 반응에 기인한 위 내막의 염증으로 발생된다. 위궤양과 십이지장궤양의 증상을 완화시키기 위하여 다양한 치료제, 예를 들면, 위에서 산 생산을 감소시키는 치료제가 항생제와 병용되고 있다. 헬리코박터 파일로리(H. pylori)에 대한 신규한 백신 역시 시도되고 있긴 하지만 제한적인 성공만이 보고되었다. 이에 더하여, 헬리코박터(Helicobacter)의 다른 종 및 다른 위장 병원균 역시 염증을 유발할 수 있다.Thus, Helicobacter pylori (H. pylori) infection is a peptic ulcer (peptic ulcer), gastric cancer, gastric mucosa - thereby increasing the risk of developing gastritis, gastric ulcer, duodenal ulcer, as well as the combined lymphoid tissue lymphoma. These problems are not directly induced by H. pylori cells but result from inflammation of the lining of the stomach due to a response to H. pylori . In order to alleviate the symptoms of gastric ulcer and duodenal ulcer, various therapeutic agents are used in combination with antibiotics, for example, those which reduce stomach acid production. Novel vaccines against H. pylori have also been tried, but only limited success has been reported. In addition, other species of Helicobacter and other gastrointestinal pathogens can also cause inflammation.
헬리코박터 파일로리(H. pylori) 감염을 연구한 최근의 한 연구 논문에서는 헬리코박터 파일로리(H. pylori) 감염이 만성 염증 반응의 일부로서 위 점액 시알릴화(sialylation)를 유도하고, 따라서 많은 독력 균주가 염증 부위에 더욱 효과적으로 부착될 수 있다고 결론하였다(Science 18:573-578, 2002). Helicobacter pylori (H. pylori) in a recent study of H. pylori infection was studied (H. pylori) infection leads to mucous above sialylation (sialylation) as part of a chronic inflammatory response, and therefore a lot of bootstrapping strains inflammation It can be concluded that it can be more effectively attached to the cell (Science 18: 573-578, 2002).
위와 위장관에서 염증은 예로써 헬리코박터 파일로리(H. pylori) 또는 다른 병원균에 의해 생성되는 항원성 자극물질에 대한 반응으로 상피에서 대식세포와 수상돌기 세포에 의해 생산되는 세포내 신호 단백질(사이토킨)에 의해 매개된다. 상피가 헬리코박터 파일로리(H. pylori) 또는 이에 의해 생성되는 내독소, 예를 들면, LPS와 같은 항원에 접촉한 직후, 상피에서 항원 제시 세포(수상돌기 세포 포함)가 이런 신호를 고유 대식세포에 전달하게 되고, 이후 대식세포는 Th-1형 반응을 유도하는데, 여기서 TNFα, IL-1, IL-6, IL-12를 비롯한 친-염증성 사이토킨이 대식세포에 의해 생산된다. 그 다음, 이들 사이토킨은 고유 킬러 세포, T-세포 및 다른 세포를 자극하여, 염증의 핵심 매개물질인 인터페론 γ(IFNγ)을 생성시킨다. IFNγ은 염증 반응 및 독성을 유발하는 상기한 반응을 고조시킨다. 또한, 고유 대식세포는 Th-2형 반응으로 항원에 반응할 수도 있다. 이들 반응은 IFNγ에 의해 억제된다. 이들 Th-2형 세포는 소염 사이토킨, 예를 들면, IL-4, IL-5, IL-9, IL-10을 생산한다.Above and in the gastrointestinal tract inflammation by pylori (H. pylori) or antigenic irritants intracellular signaling proteins (cytokines) produced by macrophages and dendritic cells in the epithelium in response to the generated by other pathogens, for example H. Is mediated. Immediately after the epithelium contacts H. pylori or endotoxins produced by it, for example, LPS, antigen presenting cells (including dendritic cells) in the epithelium transmit these signals to native macrophages. The macrophages then induce a Th-1 response, in which pro-inflammatory cytokines, including TNFα, IL-1, IL-6, IL-12, are produced by the macrophages. These cytokines then stimulate native killer cells, T-cells and other cells to produce interferon γ (IFNγ), a key mediator of inflammation. IFN [gamma] heightens the above-described reactions that cause inflammatory responses and toxicity. Intrinsic macrophages may also respond to antigens in a Th-2 type response. These responses are inhibited by IFNγ. These Th-2 type cells produce anti-inflammatory cytokines such as IL-4, IL-5, IL-9, IL-10.
IL-10은 IFNγ의 생산을 저해하고, 따라서 면역 반응을 억제하는 것으로 알려져 있다. Th-1형 세포와 Th-2형 세포간의 균형 및 이들의 개별적인 사이토킨 생산은 특정 항원에 대한 염증 반응의 정도를 결정한다. Th-2형 세포는 면역계를 통하여 면역글로불린의 생산을 촉진할 수도 있다. IFNα 수준이 감소된 위장관에서 소염 활성은 강화된 상피 세포(장벽 내막 완전성)와 상관하고, 따라서 위장 병원균과 독소에 의해 유발된 부정적인 효과를 감소시킨다.IL-10 is known to inhibit the production of IFNγ and thus inhibit the immune response. The balance between Th-1 and Th-2 cells and their individual cytokine production determine the extent of the inflammatory response to a particular antigen. Th-2 type cells may also promote the production of immunoglobulins through the immune system. Anti-inflammatory activity in the gastrointestinal tract with reduced IFNα levels correlates with enhanced epithelial cells (barrier intima integrity), thus reducing the negative effects caused by gastrointestinal pathogens and toxins.
다수의 연구 결과는 DNA가 장내 상피 세포에 소염 작용을 발휘하거나 면역계를 자극할 수 있음을 암시한다(Madsen et al. and Rachmilewitz et al, respectively, presentations at Digestive Disease Week, May 19-22, 2002, The Moscone Center, San Francisco). Many studies suggest that DNA may exert an anti-inflammatory effect on intestinal epithelial cells or stimulate the immune system (Madsen et al. And Rachmilewitz et al, respectively, presentations at Digestive Disease Week, May 19-22, 2002, The Moscone Center, San Francisco).
생쥐는 무균동물에서는 발생하지 않는 만성 대장염이 자발적으로 발병한다. 생쥐 대장염은 위장관의 만성적인 중증 염증 질환인 사람 크론병과 유사하다. 크론병은 일반적으로 장에서 발병하지만, 위장관의 다른 부위에서도 발병할 수 있다. 이들 질환은 장내 세균의 존재를 요하고 대장염의 Th1-매개된-IL-12-의존성 형태이다. 원인과 증상의 유사성으로 인하여, 생쥐 대장염 모델 및 다른 생쥐 모델은 염증 반응의 요소를 직접적으로 조사하는데 이용되는데, 사람에 동일한 기전이 적용되기 때문에 사람 위장 질환의 치료제를 개발하는데 활용될 수 있다.Mice spontaneously develop chronic colitis that does not occur in sterile animals. Mouse colitis is similar to human Crohn's disease, a chronic, severe inflammatory disease of the gastrointestinal tract. Crohn's disease usually develops in the intestine, but can also occur in other parts of the gastrointestinal tract. These diseases require the presence of enteric bacteria and are a Th1-mediated-IL-12-dependent form of colitis. Because of the similarity of causes and symptoms, the mouse colitis model and other mouse models are used to directly investigate the components of the inflammatory response, which can be used to develop a therapeutic agent for human gastrointestinal diseases because the same mechanism applies to humans.
락토바실러스 루테리(Lactobacillus reuteri)는 동물의 위장관에서 자연적으로 발생하고, 통상적으로 건강한 동물의 장 및 낮은 pH에도 불구하고 사람의 위에서 발견된다. 이는 항생 활성을 갖는 것으로 알려져 있다(U.S. Patent No. 5,439,678, 5,458,875, 5,534,253, 5,837,238, 5,849,289). 락토바실러스 루테리(L. reuteri) 세포는 글리세롤의 존재하에 혐기성 조건에서 배양되면, 루테린(β-하이드록시-프로피온알데하이드)으로 알려진 항균 물질을 생산한다. Lactobacillus reuteri occurs naturally in the gastrointestinal tract of animals and is commonly found in the stomach of humans despite the gut and low pH of healthy animals. It is known to have antibiotic activity (US Patent No. 5,439,678, 5,458,875, 5,534,253, 5,837,238, 5,849,289). L. reuteri cells, when cultured in anaerobic conditions in the presence of glycerol, produce an antimicrobial substance known as lutein (β-hydroxy-propionaldehyde).
락토바실러스 코리니포미스(L. coryniformis)는 통상적으로 사람의 구강에 존재하는 락토바실러스(Lactobacillus)의 다소 덜 알려진 종이다. 이는 토양, 비료, 식물 재료에서도 발견될 수 있다. 이는 사일리지(silage)에서 맥주 스포일러로서 발견되었고, 우수한 유산 생산 및 항곰팡이 활성이 보고되었다. 본 발명에 이용된 락토바실러스 코리니포미스(L. coryniformis) MM7 분리물(ATCC PTA-4660)은 사람의 모유에서 발견되었다. L. coryniformis is a lesser known species of Lactobacillus , which is typically present in the oral cavity of humans. It can also be found in soil, fertilizers and plant materials. It was found as a beer spoiler in silage, and excellent lactic acid production and antifungal activity was reported. L. coryniformis MM7 isolate (ATCC PTA-4660) used in the present invention was found in human breast milk.
면역조절 활성 역시 다양한 락토바실러스(Lactobacillus)와 연관한다. 여러 락토바실러스(Lactobacillus)에 의한 효과적인 항생 활성의 가능성이 알려져 있긴 하지만, 위장 염증을 감소시키는 능력에서 균간에 실질적인 차이가 존재하는 지와 이런 균주가 선별될 수 있는 지는 아직 확인되지 않고 있다.Immunomodulatory activity is also associated with a variety of Lactobacillus bacteria (Lactobacillus). Various Lactobacillus bacteria (Lactobacillus) Although the known possibility of an effective antimicrobial activity due to, but has not yet been identified in the capability of reducing gastrointestinal inflammation in which the paper and this strain to a substantial difference between the present strain may be selected.
이런 이유로, 본 발명의 목적은 위장 염증, 예를 들면, 헬리코박터 파일로리(Helicobacteri pylori)에 기인한 위장 염증을 감소시키는 능력에 기초하여 선별된 락토바실러스(Lactobacillus) 균주를 제공하는 것이다. 본 발명의 다른 목적은 사람 투여용으로 헬리코박터 파일로리(Helicobacteri pylori)와 연관된 염증의 치료 또는 예방을 위한 약품, 상기 선별된 균주가 성장하는 조정 배지, 이런 조정 배지의 단백질-함유 추출물을 비롯하여 상기 균주를 함유하는 산물을 제공하는 것이다.For this reason, it is an object of the present invention to provide Lactobacillus strains selected based on their ability to reduce gastrointestinal inflammation, eg, gastrointestinal inflammation due to Helicobacteri pylori . Another object of the invention is Helicobacter pylori (Helicobacteri pylori) adjustment to the drug, the selected strains grown for the treatment or prevention of related inflammatory and culture medium, such proteins of the conditioned medium for human administration - the strain, as well as containing extract It is to provide a product containing.
다른 목적과 이점은 아래의 상세한 설명 및 첨부된 특허청구범위로부터 더욱 명확하다.Other objects and advantages are apparent from the following detailed description and the appended claims.
본 발명은 위장 염증, 예를 들면, 헬리코박터 파일로리(Helicobacteri pylori)에 기인한 위장 염증을 감소시키는 능력에 기초하여 선별된 특정 락토바실러스(Lactobacillus) 균주 및 이런 균주를 함유하는 산물에 관하고, 또한 사람 투여용으로 헬리코박터 파일로리(Helicobacteri pylori)와 연관된 염증의 치료 또는 예방을 위한 약품, 상기 선별된 균주가 성장하는 조정 배지, 이런 조정 배지의 단백질-함유 추출물에 관한다.The present invention relates to certain Lactobacillus strains selected on the basis of their ability to reduce gastrointestinal inflammation, eg, gastrointestinal inflammation due to Helicobacteri pylori, and to products containing such strains, and also to humans. relates to extracts containing-conditioned medium, the conditioned media of this protein to the drug, the selected strains for the treatment or prevention of inflammation associated with pylori (Helicobacteri pylori) for administration Helicobacter growth.
본 발명의 다른 목적과 특징은 아래의 상세한 설명 및 첨부된 특허청구범위로부터 더욱 명확하다.Other objects and features of the present invention are more apparent from the following detailed description and the appended claims.
대식세포에 의한 자극된 친-염증성 사이토킨 생산에 강한 저해 효과를 보이는 것으로 확인된 균주는 위에서 헬리코박터 파일로리(H. pylori) 유발된 염증을 비롯하여 사람의 위장관에서 염증의 치료에 특히 효과적이다.Strains that have been shown to have a strong inhibitory effect on stimulated pro-inflammatory cytokine production by macrophages are particularly effective in the treatment of inflammation in the human gastrointestinal tract, including H. pylori induced inflammation in the stomach.
도 1은 LPS-활성화된 대식세포에 의한 TNFα 생산에 대한 락토바실러스(Lactobacillus)-조정 배지의 효과를 보여주는 막대그래프이다.
도 2는 LPS 단독에서 대식세포와 비교하여 다양한 락토바실러스(Lactobacillus) 균주와 LPS의 조정 배지의 존재하에 대식세포 TNFα 발현에서 배수적 증가를 보여주는 막대그래프이다.1 is a bar graph showing the effect of Lactobacillus -regulated medium on TNFα production by LPS-activated macrophages.
FIG. 2 is a bar graph showing a fold fold increase in macrophage TNFα expression in the presence of various Lactobacillus strains and LPS media in comparison with macrophages in LPS alone.
본 발명은 위장 염증, 예를 들면, 헬리코박터 파일로리(Helicobacteri pylori)에 기인한 위장 염증을 감소시키는 능력에 기초하여 선별된 락토바실러스(Lactobacillus) 균주에 관한다. 이런 균주는 락토바실러스 코리니포미스(Lactobacillus coryniformis) MM7, ATCC PTA-4660과 락토바실러스 루테리(Lactobacillus reuteri) MM2-3, ATCC PTA-4659이다. 이들 균주로부터 유래된 전체 세포 또는 성분을 함유하는 식품, 영양 보조제와 제제, 제약학적 또는 의학적 장치와 같은 산물은 당분야에 공지된 바와 같이 제조될 수 있고, 공지된 섭취가능 지지물 + 락토바실러스(Lactobacillus) 균주 또는 이로부터 유래된 성분을 함유한다. 우수한 TNFα 감소 능력을 갖는 것으로 이전에 확인된 균주, 예를 들면, 락토바실러스 람노수스(L. rhamnosus) GG ATCC 53103, 락토바실러스 루테리(L. reuteri) ATCC 55730 역시 상기한 제제에 이용될 수 있다. 이들 산물은 사람 투여용으로 헬리코박터 파일로리(Helicobacteri pylori)와 연관된 염증의 치료 또는 예방을 위한 약품이다.The present invention relates to Lactobacillus strains selected based on their ability to reduce gastrointestinal inflammation, eg, gastrointestinal inflammation due to Helicobacteri pylori. These strains are Lactobacillus coryniformis MM7, ATCC PTA-4660 and Lactobacillus reuteri MM2-3, ATCC PTA-4659. Products such as foods, nutritional supplements and preparations, pharmaceutical or medical devices containing whole cells or components derived from these strains can be prepared as known in the art, and known ingestible supports + Lactobacillus ) Strains or components derived therefrom. Strains previously identified as having good TNFα reducing ability, such as L. rhamnosus GG ATCC 53103, L. reuteri ATCC 55730, may also be used in the formulations described above. These products are medicines for the treatment or prevention of inflammation associated with pylori (Helicobacteri pylori) Helicobacter for human administration.
적절한 사이토킨을 이용한 모델 시스템을 활용하여 염증을 감소시키거나 증가시키는 인자를 확인한다. 본원에 기재된 실시예에서는 RAW 264.7 대식세포(ATCC, Rockville, MD, ATCC # TIB-71)를 이용한 생쥐 대식세포 분석을 활용하여 박테리아 균주, 특히 락토바실러스(Lactobacillus) 균주에서 염증 경로에 대한 효과를 조사한다. IL-10은 상기 분석에서 양성 대조로 이용되는데, IL-10 처리는 친-염증성 사이토킨, 예를 들면, TNFα(종양 괴사 인자 알파)의 저해를 보인다. 선별된 락토바실러스(Lactobacillus) 균주를 실험 배지에서 개별적으로 성장시킨 이후, 살아있는 박테리아 세포는 여과로 제거하고, 상층액(이후, “조정 배지”)은 대식세포 분석에서 검사한다. 대식세포는 먼저, 친-염증성 항원, 예를 들면, 정제된 LPS(대장균(E. coli) 유래된 리포폴리사카라이드), 포도상 구균(S. aureus) 유래된 지질타이코산(lipoteichoic acid, LTA), 또는 세포-없는 대장균(E. coli)이나 헬리코박터(Helicobacter) 조정 배지로 자극하여 TNFα를 비롯한 친-염증성 사이토킨을 생산한다. 선별된 박테리아로부터 유래된 추정 면역조절 물질을 함유하는 락토바실러스(Lactobacillus) 균주의 조정 배지는 항원-활성화된 대식세포와 동시-배양한다. 대식세포의 면역 반응을 조절하는 조정 배지의 능력은 이들 세포에 의한 TNFα 생산에서 변화로 모니터한다. 상기 분석으로부터 TNFα 프로필은 대식세포에 의한 TNFα 생산을 감소시키는데 가장 효과적인 균주의 선별을 가능하게 한다. 상기 분석 시스템에서 pH 조정에 의한 대조 실험은 변화된 pH가 관찰된 효과에 영향을 주게 되는 가능성을 제거한다. Model systems using appropriate cytokines are used to identify factors that reduce or increase inflammation. In the embodiment described herein RAW 264.7 macrophages by utilizing rat macrophages analysis using (ATCC, Rockville, MD, ATCC # TIB-71) bacterial strains, particularly Lactobacillus bacteria (Lactobacillus) investigated the effect on inflammation path from the strain do. IL-10 is used as a positive control in this assay, wherein IL-10 treatment shows inhibition of pro-inflammatory cytokines such as TNFα (tumor necrosis factor alpha). After the selected Lactobacillus strains are grown individually in the experimental medium, live bacterial cells are removed by filtration and the supernatants (hereafter "conditioned media") are examined in macrophage analysis. Macrophages first contain pro-inflammatory antigens such as purified LPS ( E. coli- derived lipopolysaccharides), Staphylococcus aureus-derived lipoteichoic acid (LTA). or cell-free by E. coli (E. coli) or H. pylori (Helicobacter) stimulated with pro-conditioned medium, including TNFα - produce inflammatory cytokines. Modulated media of Lactobacillus strains containing putative immunomodulatory substances derived from selected bacteria are co-cultured with antigen-activated macrophages. The ability of regulatory media to modulate the immune response of macrophages is monitored by changes in TNFα production by these cells. The TNFα profile from this assay allows the selection of strains that are most effective at reducing TNFα production by macrophages. Control experiments with pH adjustment in the assay system eliminate the possibility that the changed pH will affect the observed effect.
놀랍게도, 매우 유사한 사람으로부터 유래된 겉보기에 유사한 락토바실러스(Lactobacillus)의 박테리아 분리물과 균주는 친-염증성 항원에 대한 반응에 기인한 대식세포에 의한 TNFα의 생산에 매우 가변적이고 차별적인 영향을 준다. 이들 균주는 유전자 핑거프린팅(genetic fingerprinting)으로도 구별할 수 없는데, 그 이유는 이들이 최대 98%까지 유전적으로 유사하면서도 면역 세포에 매우 차별적인 효과를 보이기 때문이다. 따라서, 대식세포에 의한 자극된 친-염증성 사이토킨 생산에 강한 저해 효과를 보이는 것으로 확인된 균주는 위에서 헬리코박터 파일로리(H. pylori) 유발된 염증을 비롯하여 사람의 위장관에서 염증의 치료에 특히 효과적이다.Surprisingly, apparently similar bacterial isolates and strains of Lactobacillus from very similar humans have a highly variable and differential effect on the production of TNFα by macrophages due to responses to pro-inflammatory antigens. These strains are indistinguishable even by genetic fingerprinting, because they are genetically similar up to 98%, but have very different effects on immune cells. Thus, strains that have been shown to have a strong inhibitory effect on stimulated pro-inflammatory cytokine production by macrophages are particularly effective in the treatment of inflammation in the gastrointestinal tract of humans, including H. pylori induced inflammation in the stomach.
본 발명의 특징은 아래의 실시예를 참고하면 더욱 분명하지만, 이들 실시예는 본 발명을 한정하지 않는다.Features of the present invention are more apparent with reference to the following examples, but these examples do not limit the present invention.
실시예 1. 소염 균주의 선별.Example 1. Screening of anti-inflammatory strains.
락토바실러스 종(Lactobacillus spp)(예로써 락토바실러스 람노수스(L. rhamnosus) GG ATCC 53103, 락토바실러스 존소닐(L. johnsonii) ATCC 33200, 락토바실러스 루테리(Lactobacillus reuteri) MM2-3 ATCC PTA-4659, 락토바실러스 코리니포미스(L. coryniformis) MM7, ATCC PTA-4660 포함) 및 대장균(E coli) Nissle은 각각 de Man, Rogosa, Sharpe(MRS)와 Luria-Bertani(LB) 배지(Difco, Sparks, MD)에 배양하였다. 락토바실러스(Lactobacillus)의 하룻밤 배양액은 1.0의 OD600(대략 109 세포/㎖)로 희석하고 1:1O 희석하며 추가로 4시간, 8시간, 24시간동안 배양하였다. 헬리코박터 파일로리(Helicobacteri pylori)(Sydney 균주 SS1)와 헬리코박터 헤파티쿠스(Helicobacter hepaticus) 3B1(ATCC 51449)은 10% 소 태아 혈청(FBS)으로 보충된 Brucella 액체 배지(Difco)에서 48시간동안 배양하였다. 배양액은 1:1O 희석하고 추가로 24시간과 48시간동안 배양하였다. 박테리아 세포-없는 조정 배지는 4℃에서 8500 rpm으로 10분동안 원심분리하여 수집하였다. 조정 배지는 세포 펠렛으로부터 분리하고 0.22 ㎛ 세공 필터 단위(Millipore, Bedford, Mass.)에 여과하였다. Lactobacillus spp (e.g. L. rhamnosus GG ATCC 53103, L. johnsonii ATCC 33200, Lactobacillus reuteri MM2-3 ATCC PTA-4659, L. coryniformis MM7, including ATCC PTA-4660 and E coli Nissle were de Man, Rogosa, Sharpe (MRS) and Luria-Bertani (LB) media (Difco, Sparks, Incubated in MD). Overnight cultures of Lactobacillus were diluted with an OD600 of 1.0 (approximately 10 9 cells / ml), diluted 1: 1O and incubated for an additional 4 hours, 8 hours, and 24 hours. Helicobacter pylori (Helicobacteri pylori) (Sydney strain SS1) and H. H. party kusu (Helicobacter hepaticus) 3B1 (ATCC 51449 ) was cultured in the Brucella broth (Difco) supplemented with 10% fetal bovine serum (FBS) for 48 hours. The culture was diluted 1: 1O and incubated for an additional 24 and 48 hours. Bacterial cell-free conditioned media was collected by centrifugation at 8500 rpm for 10 minutes at 4 ° C. The conditioned medium was separated from the cell pellet and filtered in a 0.22 μm pore filter unit (Millipore, Bedford, Mass.).
생쥐 단핵구/대식세포 세포주, RAW 264.7(ATCC TIB-71)과 RAW 264.7 감마 NO(-)(ATCC CRL-2278)는 염증 반응 경로를 조사하기 위한 리포터 세포로 이용하였다. RAW 264.7 세포는 80-90% 합류 수준까지 5% C02, 37℃에서 10% FBS와 2% 항생제(5000 units/㎖ 페니실린과 5 ㎎/㎖ 스트렙토마이신, Sigma)로 보충된 Dulbecco's Modified Eagle Medium(야생형) 또는 RPM1 Medium 1640(감마 NO-)(GibcoInvitrogen, Carlsbad, CA)에 배양하였다. 대략 5 x 104 세포를 96-웰 세포 배양액 클러스터에 접종하고, 리포폴리사카라이드(LPS) 활성화와 조정 배지의 첨가에 앞서 2시간동안 부착시켰다. 고유 RAW 264.7 세포는 대장균(E. coli) 혈청형 0127:B8(Sigma)의 정제된 LPS에 노출시켰다. 활성화 배지는 2 ng LPS를 20 ㎕ 조정 배지/웰에 첨가하여 만들었다. 대식세포는 미리-배양하거나 세포-없는 락토바실러스(Lactobacillus) 조정 배지와 함께 동시-배양하였다. 재조합 mIL-10(R&D Systems, Minneapolis, Min.)은 양성 대조로 이용하였다. 세포 생존능은 트립탄-블루(Invitrogen) 배제로 사정하였다. 세포 배양 상층액에서 TNF-α의 존재는 샌드위치 효소 면역분석법, Quantikine M® Mouse TNF-α Immunoassay(R & D Systems)로 측정하였다. Mouse monocyte / macrophage cell lines, RAW 264.7 (ATCC TIB-71) and RAW 264.7 gamma NO (-) (ATCC CRL-2278), were used as reporter cells to investigate the inflammatory response pathway. RAW 264.7 cells to 80-90% confluence level supplemented in 5% C0 2, 37 ℃ with 10% FBS and 2% antibiotic (5000 units / ㎖ penicillin and 5 ㎎ / ㎖ streptomycin, Sigma) Dulbecco's Modified Eagle Medium ( Wild type) or RPM1 Medium 1640 (gamma NO-) (Gibco Invitrogen, Carlsbad, Calif.). Approximately 5 × 10 4 cells were seeded in 96-well cell culture clusters and allowed to attach for 2 hours prior to lipopolysaccharide (LPS) activation and addition of conditioned media. Native RAW 264.7 cells were exposed to purified LPS of E. coli serotype 0127: B8 (Sigma). Activation medium was made by adding 2 ng LPS to 20 μl conditioned medium / well. Macrophages were co-cultured with pre-cultured or cell-free Lactobacillus conditioned media. Recombinant mIL-10 (R & D Systems, Minneapolis, Min.) Was used as a positive control. Cell viability was assessed by triptan-blue (Invitrogen) exclusion. The presence of TNF-α in cell culture supernatants was measured by sandwich enzyme immunoassay, Quantikine M® Mouse TNF-α Immunoassay (R & D Systems).
LPS-활성화된 대식세포에 의한 TNFα 생산에 대한 락토바실러스(Lactobacillus)-조정 배지의 효과는 도 1에 도시하는데, 이는 검사된 45가지 균주 중에서 여러 상이한 균주가 활성화된 대식세포에 의한 TNFα 생산을 감소시킬 수 있음을 입증한다. 도 2에서는 LPS 단독과 비교하여 다양한 락토바실러스(Lactobacillus) 균주에서 TNFα 발현의 배수적 변화를 보여준다. 이후, 이들 연구 결과를 이용하여 가장 효과적인 균주를 선별한다. 도면에서는 언급되었지만 명세서에서 구체적으로 언급되지 않은 균주는 검사된 락토바실러스(Lactobacillus) 균주, 특히 락토바실러스 루테리(L. reuteri)이다.The effect of Lactobacillus -modulated medium on TNFα production by LPS-activated macrophages is shown in FIG. 1, which reduces TNFα production by activated macrophages among several 45 strains tested. Prove that you can. Figure 2 shows a fold change in TNFα expression in various Lactobacillus strains compared to LPS alone. The results of these studies are then used to select the most effective strains. Strains mentioned in the figures but not specifically mentioned in the specification are tested Lactobacillus strains, in particular L. reuteri .
본 실시예에서는 상기한 방법을 이용하여 표준 요구르트 첨가용으로 락토바실러스 코리니포미스(L. coryniformis) MM7, ATCC PTA-4660을 선별하였다. 상기 락토바실러스 코리니포미스(L. coryniformis) 균주는 유가공 산업에서 락토바실러스(Lactobacillus)를 성장시키는 표준 방법을 이용하여 배양하고 동결 건조시켰다. 이후, 상기 배양액은 전통적인 요구르트 배양액을 이용하여 발효된 우유에 10E+7 CFU/요구르트 g의 수준으로 첨가되는데, 상기 요구르트는 헬리코박터 파일로리(Helicobacteri pylori)에 의해 유발된 위염의 예방제로서 사람에 사용되었다.In this example, L. coryniformis MM7 and ATCC PTA-4660 were selected for the addition of standard yoghurt using the method described above. The L. coryniformis strain was cultured and lyophilized using a standard method of growing Lactobacillus in the dairy industry. The culture was then added to the fermented milk using traditional yogurt culture at a level of 10E + 7 CFU / yogurt g, which was used in humans as a preventive of gastritis caused by Helicobacteri pylori .
실시예 2: 조정 배지의 이용Example 2: Use of Conditioning Medium
본 실시예에서는 상기한 방법을 이용하여 효과적인 TNFα 감소 균주, 특히 락토바실러스 루테리(L. reuteri) ATCC PTA-4659의 조정 배지를 선별하였다. 상기 배지는 de Man, Rogosa, Sharpe(MRS)(Difco, Sparks, MD)에서 상기 균주를 배양함으로써 대규모로 생산하였다. 락토바실러스(Lactobacillus)의 하룻밤 배양액은 1.0의 OD600(대략 109 세포/㎖)로 희석하고 1:1O 희석하며 추가로 24시간동안 배양하였다. 박테리아-세포 없는 조정 배지는 4℃에서, 8500 rpm으로 10분동안 원심분리하여 수집하였다. 조정 배지는 세포 펠렛으로부터 분리하고 0.22 ㎛ 세공 필터 단위(Millipore, Bedford, Mass.)에 여과하였다. 이후, 조정 배지는 표준 방법을 이용하여 동결 건조시키고 정제로 제조하였다. 상기 정제는 헬리코박터 파일로리(H. pylori)에 의해 유발된 궤양을 치료하는 약물로서 사람에 사용되었다. In this example, the above-described method was used to select effective TNFα reducing strains, in particular, L. reuteri ATCC PTA-4659 conditioned medium. The medium was produced on a large scale by culturing the strain in de Man, Rogosa, Sharpe (MRS) (Difco, Sparks, MD). Overnight cultures of Lactobacillus were diluted with an OD600 of 1.0 (approximately 10 9 cells / ml), diluted 1: 1O and incubated for an additional 24 hours. Bacterial-cell free conditioned media was collected by centrifugation at 8500 rpm for 10 minutes at 4 ° C. The conditioned medium was separated from the cell pellet and filtered in a 0.22 μm pore filter unit (Millipore, Bedford, Mass.). The conditioned medium was then lyophilized using standard methods and made into tablets. This tablet has been used in humans as a drug to treat ulcers caused by H. pylori .
실시예 3. 락토바실러스 루테리( Lactobacillus reuteri ) 균주의 DNA-핑거프린팅 Example 3. DNA-fingerprinting of Lactobacillus reuteri strain
U.S. Patent No. 5,523,217과 5,691,136((Lupski et al.)의 방법을 이용하여 락토바실러스 루테리(L. reuteri) 균주의 게놈 핑거프린팅을 실행하였다. 상기 방법은 한 쌍의 외향 프라이머를 박테리아 시료에 첨가함으로써 박테리아 DNA의 증폭을 이용한다. 증폭이후, 혼성화에 의한 신장 산물은 크기로 분리하고, 박테리아 균주는 크기에 따라 분류된 신장 산물의 패턴을 측정함으로써 특성화한다. Uprime E 프라이머(1개의 프라이머)를 이용하여 Bacterial BarCodes, Inc.(Houston, TX)의 82가지 락토바실러스 루테리(L. reuteri) 균주에 대한 이중 겔 영상을 얻었다. 이중의 데이터 세트는 동등하였다. 서로 구별되는 총 11개의 클러스터 및 독특한 8개의 분리물이 존재하였다.US Patent No. Genome fingerprinting of L. reuteri strains was performed using the methods of 5,523,217 and 5,691,136 ((Lupski et al.) The method amplifies bacterial DNA by adding a pair of extroverted primers to the bacterial sample. After amplification, kidney products by hybridization are separated by size, and bacterial strains are characterized by measuring the pattern of kidney products sorted by size Bacterial BarCodes, Inc. using Uprime E primer (one primer). Dual gel images of 82 L. reuteri strains of., Houston, TX, were obtained, the data sets being equivalent, there were a total of 11 distinct clusters and 8 unique isolates. .
TNF-α를 감소시키는데 효과적인 것으로 확인된 균주(도 1과 2)는 상기 방법에서 일군으로 분류되지 않는데, 이는 TNFα 감소 능력을 가진 여러 균주를 발견하기 위하여 DNA-핑거프린팅을 이런 방식으로 이용하는 것이 충분하지 않음을 입증한다.Strains found to be effective in reducing TNF-α (FIGS. 1 and 2) are not grouped in this method, which is sufficient to use DNA-fingerprinting in this way to find several strains with TNFα reduction ability. Prove it doesn't.
실시예 4. 효과적인 락토바실러스( Lactobacillus ) 균주에 의해 생산된 단백질의 특성화 Example 4 Characterization of Proteins Produced by Effective Lactobacillus Strains
락토바실러스 루테리(L. reuteri) 균주 MM2-3 조정 배지를 비롯한 서로 다른 효과적인 락토바실러스(Lactobacillus) 조정 배지는 다양한 변성 화합물로 처리하여 박테리아로부터 유래된 추정 면역모듈린(immunomodulin)의 특성을 결정하였다. 따라서, 조정 배지는 반복적 냉동-해동, 가열 처리, DNA 절단 효소로 절단, 프로테아제, 비활성화된 프로테아제를 가하였다. 이런 방식으로, 추정 면역모듈린은 성질에서 한가지이상의 단백질 또는 펩티드인 것으로 확인되었다. 추정 단백질 면역모듈린의 크기를 결정하기 위하여, 조정 배지는 여과로 단편화시키고, 여과액은 효능을 검사하였다. 이런 방식으로, 효과적인 락토바실러스(Lactobacillus) 균주의 조정 배지의 활성 성분은 크기가 대략 5 kDa 이하인 것으로 밝혀졌다. The characteristics of Lactobacillus ruteri (L. reuteri) strain MM2-3 Different effective Lactobacillus bacteria (Lactobacillus) conditioned medium, including the conditioned medium by treatment with a variety of modified compound derived from bacteria module estimates immune Lin (immunomodulin) was determined. Thus, the conditioned medium was subjected to repeated freeze-thaw, heat treatment, digestion with DNA cleavage enzyme, protease, inactivated protease. In this way, the putative immunomodulin has been identified as one or more proteins or peptides in nature. To determine the size of putative protein immunomodulin, conditioned media was fragmented by filtration and the filtrate was tested for efficacy. In this way, it was found that the active ingredient of the conditioned medium of an effective Lactobacillus strain is approximately 5 kDa or less in size.
본 발명은 특정 구체예를 참조하여 기술하였지만, 다수의 개변이 가능하고, 따라서 이런 모든 개변은 본 발명의 기술적 사상과 범위에 속하는 것으로 간주된다Although the present invention has been described with reference to specific embodiments, many modifications are possible and all such modifications are considered to be within the spirit and scope of the invention.
Claims (4)
a) 락토바실러스(Lactobacillus) 균주들만을 선별하여 배양하고;
b) 각 균주로부터 무세포(cell free) 배양 상청액을 수득하고;
c) 상청액에 배양된 각 세포의 TNF알파 생산에서 상청액의 TNF알파 생산에 효과를 평가하고, 이때 기준 시료와 비교하여 각 세포에서 TNF알파 생산이 감소되는 젖산 균주를 확인하고; 그리고
d) TNF알파 생산이 감소된 해당 젖산 균주만을 선택하는 단계로 구성된 방법.In the screening methods of lactic acid strains for administration to a mammal to within the mammalian gastrointestinal Helicobacter pylori (Helicobacter pylori) to reduce gastrointestinal inflammation associated with infection,
a) selecting and culturing only Lactobacillus strains;
b) obtaining cell free culture supernatant from each strain;
c) assessing the effect on TNFalpha production of supernatant in the TNFalpha production of each cell cultured in the supernatant, identifying lactic acid strains in which TNFalpha production is reduced in each cell compared to the reference sample; And
d) selecting only those lactic acid strains with reduced TNFalpha production.
a. 제 3 항에 따른 락토바실러스(Lactobacillus) 균주를 선별하고;
b. 상기 균주로부터 소염 성분을 수득하고;
c. 상기 성분을 섭취가능 지지물에 첨가하여 식품을 제공한다.A process for preparing a food composition, comprising the following steps:
a. Selecting a Lactobacillus strain according to claim 3;
b. Obtaining an anti-inflammatory component from said strain;
c. The ingredient is added to the ingestible support to provide food.
a. 제 3 항에 따른 락토바실러스(Lactobacillus) 균주를 선별하고;
b. 상기 균주로부터 소염 성분을 수득하고;
c. 상기 성분을 제약학적 담체에 첨가하여 제약학적 조성물을 제공한다.A method for preparing a pharmaceutical composition, comprising the following steps:
a. Selecting a Lactobacillus strain according to claim 3;
b. Obtaining an anti-inflammatory component from said strain;
c. The ingredient is added to a pharmaceutical carrier to provide a pharmaceutical composition.
a. 제 3 항에 따른 락토바실러스(Lactobacillus) 균주를 선별하고;
b. 상기 균주로부터 소염 성분을 수득하고;
c. 상기 성분을 섭취가능 지지물에 첨가하여 영양 보조제를 제공한다.
A method of making a nutritional supplement, comprising the following steps:
a. Selecting a Lactobacillus strain according to claim 3;
b. Obtaining an anti-inflammatory component from said strain;
c. The ingredient is added to the ingestible support to provide a nutritional supplement.
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US7105336B2 (en) * | 2002-10-07 | 2006-09-12 | Biogaia Ab | Selection and use of lactic acid bacteria for reducing inflammation caused by Helicobacter |
WO2004034808A1 (en) * | 2002-10-18 | 2004-04-29 | Biogaia Ab | Method of improving immune function in mammals using lactobacillus reuteri strains |
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EP1551952A1 (en) | 2005-07-13 |
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JP2006501823A (en) | 2006-01-19 |
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KR101064058B1 (en) | 2011-09-08 |
AU2003269756B2 (en) | 2007-08-09 |
CN1694954A (en) | 2005-11-09 |
KR20050047107A (en) | 2005-05-19 |
JP5439315B2 (en) | 2014-03-12 |
CN1694954B (en) | 2010-05-12 |
JP2011021015A (en) | 2011-02-03 |
WO2004031368A1 (en) | 2004-04-15 |
ES2602679T3 (en) | 2017-02-22 |
US7105336B2 (en) | 2006-09-12 |
JP4804755B2 (en) | 2011-11-02 |
BR0314511A (en) | 2005-08-09 |
MXPA05003731A (en) | 2005-06-17 |
US20060251636A1 (en) | 2006-11-09 |
HUE031627T2 (en) | 2017-09-28 |
AU2003269756A1 (en) | 2004-04-23 |
US20040067573A1 (en) | 2004-04-08 |
US7678553B2 (en) | 2010-03-16 |
HK1079811A1 (en) | 2006-04-13 |
US7960137B2 (en) | 2011-06-14 |
US20100129337A1 (en) | 2010-05-27 |
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